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RhoB regulates cell migration through altered focal adhesion dynamics.

Vega FM, Colomba A, Reymond N, Thomas M, Ridley AJ - Open Biol (2012)

Bottom Line: RhoB-depleted cells migrate faster, but less persistently in a chemotactic gradient, and frequently round up during migration.They have lower levels of surface β1 integrin, and β1 integrin activity is reduced in actin-rich protrusions.We propose that RhoB contributes to directional cell migration by regulating β1 integrin surface levels and activity, thereby stabilizing lamellipodial protrusions.

View Article: PubMed Central - PubMed

Affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, New Hunt's House, Guy's Campus, London SE1 1UL, UK.

ABSTRACT
The Rho GTPase RhoB has been shown to affect cell migration, but how it does this is not clear. Here we show that cells depleted of RhoB by RNAi are rounded and have defects in Rac-mediated spreading and lamellipodium extension, although they have active membrane ruffling around the periphery. Depletion of the exchange factor GEF-H1 induces a similar phenotype. RhoB-depleted cells migrate faster, but less persistently in a chemotactic gradient, and frequently round up during migration. RhoB-depleted cells have similar numbers of focal adhesions to control cells during spreading and migration, but show more diffuse and patchy contact with the substratum. They have lower levels of surface β1 integrin, and β1 integrin activity is reduced in actin-rich protrusions. We propose that RhoB contributes to directional cell migration by regulating β1 integrin surface levels and activity, thereby stabilizing lamellipodial protrusions.

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RhoB affects cell migration speed and directionality. (a) Migration speed of siRNA-transfected PC3 cells on tissue culture-treated plastic. n > 50 cells per condition from three different experiments. Results with two different siRNAs for RhoB and two siControl oligos are pooled; cells were tracked for 12–14 h, 58–60 h after transfection. (b) Montage from the electronic supplementary material, movie S3 showing representative GFP-actin-expressing PC3 cells. Tracks panels (right) show movement of the shown cells over time. Scale bars, 20 µm. (c) Forward migration index (FMI) and directionality of siRNA-transfected PC3 cells in a chemotaxis assay. n > 200 cells per condition from four different experiments using two different siRNAs for RhoB and two siControl oligos. Boxes of box and whisker plots show median, 25th and 75th percentile; whiskers show 95th percentile; ***p < 0.001. Rose plots show the proportion of cells with a migration direction lying within each 20° interval. Red arrow represents mean direction of cell migration; green segment, 95% CI.
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RSOB120076F4: RhoB affects cell migration speed and directionality. (a) Migration speed of siRNA-transfected PC3 cells on tissue culture-treated plastic. n > 50 cells per condition from three different experiments. Results with two different siRNAs for RhoB and two siControl oligos are pooled; cells were tracked for 12–14 h, 58–60 h after transfection. (b) Montage from the electronic supplementary material, movie S3 showing representative GFP-actin-expressing PC3 cells. Tracks panels (right) show movement of the shown cells over time. Scale bars, 20 µm. (c) Forward migration index (FMI) and directionality of siRNA-transfected PC3 cells in a chemotaxis assay. n > 200 cells per condition from four different experiments using two different siRNAs for RhoB and two siControl oligos. Boxes of box and whisker plots show median, 25th and 75th percentile; whiskers show 95th percentile; ***p < 0.001. Rose plots show the proportion of cells with a migration direction lying within each 20° interval. Red arrow represents mean direction of cell migration; green segment, 95% CI.

Mentions: The dramatic change in morphology and lamellipodial dynamics induced by RhoB knockdown suggested it would affect cell migration. RhoB-depleted PC3 cells showed a significant increase in migration speed both on uncoated and Matrigel-coated surfaces (see figure 4a; electronic supplementary material, movie S2). They also moved less persistently. This contrasts with our observations that RhoA or RhoC depletion reduces migration speed under similar conditions [8]. Cell tracking indicated that RhoB-depleted cells could extend a single small protrusion, polarize and migrate in one direction for a short time, but often went into a ‘tumbling’ phase when they extended multiple small protrusions in different directions, and then chose a different one to form a new leading edge (see figure 4b; electronic supplementary material, movies S3). By contrast, control cells had much broader and more persistent lamellipodia. As a result, RhoB-depleted cells moved faster, but with frequent turns. Similar migratory behaviour was observed in randomly migrating LnCAP or single DU145 cells (not shown).Figure 4.


RhoB regulates cell migration through altered focal adhesion dynamics.

Vega FM, Colomba A, Reymond N, Thomas M, Ridley AJ - Open Biol (2012)

RhoB affects cell migration speed and directionality. (a) Migration speed of siRNA-transfected PC3 cells on tissue culture-treated plastic. n > 50 cells per condition from three different experiments. Results with two different siRNAs for RhoB and two siControl oligos are pooled; cells were tracked for 12–14 h, 58–60 h after transfection. (b) Montage from the electronic supplementary material, movie S3 showing representative GFP-actin-expressing PC3 cells. Tracks panels (right) show movement of the shown cells over time. Scale bars, 20 µm. (c) Forward migration index (FMI) and directionality of siRNA-transfected PC3 cells in a chemotaxis assay. n > 200 cells per condition from four different experiments using two different siRNAs for RhoB and two siControl oligos. Boxes of box and whisker plots show median, 25th and 75th percentile; whiskers show 95th percentile; ***p < 0.001. Rose plots show the proportion of cells with a migration direction lying within each 20° interval. Red arrow represents mean direction of cell migration; green segment, 95% CI.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3376739&req=5

RSOB120076F4: RhoB affects cell migration speed and directionality. (a) Migration speed of siRNA-transfected PC3 cells on tissue culture-treated plastic. n > 50 cells per condition from three different experiments. Results with two different siRNAs for RhoB and two siControl oligos are pooled; cells were tracked for 12–14 h, 58–60 h after transfection. (b) Montage from the electronic supplementary material, movie S3 showing representative GFP-actin-expressing PC3 cells. Tracks panels (right) show movement of the shown cells over time. Scale bars, 20 µm. (c) Forward migration index (FMI) and directionality of siRNA-transfected PC3 cells in a chemotaxis assay. n > 200 cells per condition from four different experiments using two different siRNAs for RhoB and two siControl oligos. Boxes of box and whisker plots show median, 25th and 75th percentile; whiskers show 95th percentile; ***p < 0.001. Rose plots show the proportion of cells with a migration direction lying within each 20° interval. Red arrow represents mean direction of cell migration; green segment, 95% CI.
Mentions: The dramatic change in morphology and lamellipodial dynamics induced by RhoB knockdown suggested it would affect cell migration. RhoB-depleted PC3 cells showed a significant increase in migration speed both on uncoated and Matrigel-coated surfaces (see figure 4a; electronic supplementary material, movie S2). They also moved less persistently. This contrasts with our observations that RhoA or RhoC depletion reduces migration speed under similar conditions [8]. Cell tracking indicated that RhoB-depleted cells could extend a single small protrusion, polarize and migrate in one direction for a short time, but often went into a ‘tumbling’ phase when they extended multiple small protrusions in different directions, and then chose a different one to form a new leading edge (see figure 4b; electronic supplementary material, movies S3). By contrast, control cells had much broader and more persistent lamellipodia. As a result, RhoB-depleted cells moved faster, but with frequent turns. Similar migratory behaviour was observed in randomly migrating LnCAP or single DU145 cells (not shown).Figure 4.

Bottom Line: RhoB-depleted cells migrate faster, but less persistently in a chemotactic gradient, and frequently round up during migration.They have lower levels of surface β1 integrin, and β1 integrin activity is reduced in actin-rich protrusions.We propose that RhoB contributes to directional cell migration by regulating β1 integrin surface levels and activity, thereby stabilizing lamellipodial protrusions.

View Article: PubMed Central - PubMed

Affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, New Hunt's House, Guy's Campus, London SE1 1UL, UK.

ABSTRACT
The Rho GTPase RhoB has been shown to affect cell migration, but how it does this is not clear. Here we show that cells depleted of RhoB by RNAi are rounded and have defects in Rac-mediated spreading and lamellipodium extension, although they have active membrane ruffling around the periphery. Depletion of the exchange factor GEF-H1 induces a similar phenotype. RhoB-depleted cells migrate faster, but less persistently in a chemotactic gradient, and frequently round up during migration. RhoB-depleted cells have similar numbers of focal adhesions to control cells during spreading and migration, but show more diffuse and patchy contact with the substratum. They have lower levels of surface β1 integrin, and β1 integrin activity is reduced in actin-rich protrusions. We propose that RhoB contributes to directional cell migration by regulating β1 integrin surface levels and activity, thereby stabilizing lamellipodial protrusions.

Show MeSH
Related in: MedlinePlus